Thalamic Network Oscillations Synchronize ...

Cerebral Cortex June 2013;23:1299–1316 doi:10.1093/cercor/bhs103 Advance Access publication May 16, 2012

Thalamic Network Oscillations Synchronize Ontogenetic Columns in the Newborn Rat Barrel Cortex Jenq-Wei Yang1, , Shuming An1, Jyh-Jang Sun1, Vicente Reyes-Puerta1, Jennifer Kindler1, Thomas Berger1,2, Werner Kilb1 and Heiko J. Luhmann1 1

Institute of Physiology and Pathophysiology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany and 2Current address: Institute of Physiology, University of Bern, Bern, Switzerland

Address correspondence to Heiko J. Luhmann, Institute of Physiology and Pathophysiology, University Medical Center, Johannes Gutenberg University Mainz, Duesbergweg 6, D-55128 Mainz, Germany. Email: [email protected]

J.W.Y. and S.A. contributed equally to this work.

Keywords: activity dependent, columnar organization, development, in vivo, newborn rat Introduction A hallmark of the neocortical architecture is the structural and functional organization in radial units termed cortical columns (Mountcastle 1957; Hubel and Wiesel 1962). In many sensory and motor cortical areas, a cortical column represents the basic module for processing of afferent, intrinsic, and efferent neuronal information (Mountcastle 1997; Jones and Rakic 2010). Cortical columns vary from 300 to 600 µm in diameter depending on the species, cortical area, and on the animal’s age and undergo activity- and experience-dependent structural and functional modifications during critical periods (Hensch 2005; Hooks and Chen 2007). A rudimentary pattern of columnar units develops already during early corticogenesis. The “radial unit hypothesis” predicts that progenitors in the ventricular zone of the embryonic cortex produce cohorts of postmitotic neurons that migrate in an insidefirst-outside-last pattern along the radial glial cells into the developing cortex forming the early, so-called ontogenetic columns (Rakic 1988; Rakic et al. 2009). This hypothesis has been supported by a number of recent studies demonstrating that proliferative radial glial cells represent the neuronal progenitors (Fishell and Kriegstein 2003) and that clonally related neurons form columnar units in the developing © The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: [email protected]

(Noctor et al. 2001) and mature cortex (Yu et al. 2009). Disturbances in this early development of the columnar architecture may cause disorders in cortical processing associated with neurological deficits such as autism (Amaral et al. 2008) or Fragile X syndrome associated with mental retardation (Bureau et al. 2008; Bureau 2009). Imaging of intrinsic optical signals and single-unit microelectrode recordings in the primary visual cortex of developing cats have shown that the basic structure of the cortical map for orientation and ocular dominance columns is present at birth and does not depend on visual experience (Crair et al. 1998). Anatomical tracer studies in the visual cortex of newborn ferrets have demonstrated a surprisingly early and rapid development of ocular dominance columns (Crowley and Katz 2000), indicating that molecular cues and/or early neuronal activity guide the initial formation of cortical columns (Katz and Crowley 2002). Neuronal domains of spontaneously coactive neurons coupled through gap junctions and organized in a columnar manner have been demonstrated in the somatosensory cortex of newborn rats in slices (Yuste et al. 1992; Sun and Luhmann 2007) and in intact in vitro preparations of the cerebral cortex (Dupont et al. 2006). However, the role of the neuronal activity in shaping these early columnar networks is not understood. An intrinsic patchy organization of correlated spontaneous activity with a periodicity of ∼1 mm, which is in direct relationship with the pattern of early ocular dominance columns, has been demonstrated in the developing visual cortex of ferrets before eye-opening (Chiu and Weliky 2001, 2002). Another source for driving the initial patterning of cortical columnar circuits is the thalamus and the thalamocortical loop. Multi-electrode recordings in the lateral geniculate nucleus of young ferrets revealed patterns of spontaneous activity in the thalamus before eye-opening (Weliky and Katz 1999). Furthermore, a recent study shows that early gamma oscillations enable precise spatiotemporal thalamocortical synchronization in the neonatal rat ( postnatal day [P] P2–P7) whisker sensory system (Minlebaev et al. 2011). However, it is not clear to which extent this intrathalamic activity contributes to the generation of spontaneous cortical patterns and the development of functional ontogenetic columns, particularly at an age when cortical layers are still formed (P0–P1 in rats). It is also not known whether spontaneous activity patterns recorded during the early stages of corticogenesis resemble in their spatiotemporal characteristics sensory-evoked responses or whether spontaneous- and sensory-evoked activities represent 2 different entities of the developing cortical network.

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Neocortical areas are organized in columns, which form the basic structural and functional modules of intracortical information processing. Using voltage-sensitive dye imaging and simultaneous multi-channel extracellular recordings in the barrel cortex of newborn rats in vivo, we found that spontaneously occurring and whisker stimulation-induced gamma bursts followed by longer lasting spindle bursts were topographically organized in functional cortical columns already at the day of birth. Gamma bursts synchronized a cortical network of 300–400 µm in diameter and were coherent with gamma activity recorded simultaneously in the thalamic ventral posterior medial (VPM) nucleus. Cortical gamma bursts could be elicited by focal electrical stimulation of the VPM. Whisker stimulation-induced spindle and gamma bursts and the majority of spontaneously occurring events were profoundly reduced by the local inactivation of the VPM, indicating that the thalamus is important to generate these activity patterns. Furthermore, inactivation of the barrel cortex with lidocaine reduced the gamma activity in the thalamus, suggesting that a cortico-thalamic feedback loop modulates this early thalamic network activity.

Material and Methods Surgical Preparation All experiments were conducted in accordance with the national laws for the use of animals in research and approved by the local ethical committee (#23177-07/G10-1-010). In total 95 pups from 65 litters were investigated. VSDI, field potential (FP), and multiple-unit activity (MUA) recordings were performed in the barrel cortex of P0–P7 Wistar rats using experimental protocols similar as described previously (Yang et al. 2009) (Supplementary Fig. S1). Under deep hypothermia combined with an initial light intraperitoneal urethane anesthesia (1 g/kg, Sigma-Aldrich, Taufkirchen, Germany), the animal’s head was fixed into the stereotaxic apparatus using one aluminum holder fixed with dental cement on the occipital bones. Depending on the experimental design, the skull was opened in 1 of 2 different ways: 1) For VSDI experiments, a 3 × 3 mm2 area of the skull (0–3 mm posterior to bregma and 1.5–4.5 mm from the midline) was thinned on the left hemisphere using a miniature drill until the residual bone, but not the dura mater, above the barrel cortex could be carefully removed with a fine canula and 2) for experiments performing simultaneous multi-channel extracellular electrophysiological recordings in both the thalamus and barrel cortex, a 2 × 2 mm2 area of

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the skull (in P0–P1 animals: 0–2 mm posterior to bregma and 1.5–3.5 mm from the midline) above the barrel cortex of the left hemisphere was exposed. Additionally another rectangular area (P0–P1: 1–2.5 mm posterior to bregma and 1–2 mm from the midline) of the skull was removed for perpendicular insertion of the thalamic multi-channel electrode. One silver wire was inserted into the cerebellum and served as a ground electrode. The animals were kept at a constant temperature of 37°C by placing them on a heating blanket and covering their bodies with cotton. This experimental set-up allowed VSDI and multi-electrode electrophysiological recordings in light urethane anesthetized animals (6 ( pre-)barrel-related columns. The number of animals/litters investigated for in each age group is given in parentheses. Statistical significant differences (Mann–Whitney–Wilcoxon test) versus the P0 age group are indicated by *P < 0.05.

electrophysiological response to C2-whisker stimulation also consisted of a rather localized activity (Fig. 3A2, right). When all 8-shank recording electrodes (S1–S8) were positioned in an appropriate manner, the stimulus-evoked Cerebral Cortex June 2013, V 23 N 6 1303


most significant differences in the evoked cortical activity patterns (Fig. 1E–G). In P0–P1 rats, spontaneous events occurred every 4.9 ± 1.7 s and revealed a shorter average duration at the half-maximal amplitude of 247.6 ± 6.4 ms (P < 0.05, n = 317 events recorded in 5 P0–P1 rats from 5 litters) when compared with the evoked responses (300.8 ± 23.6 ms, n = 15 events in the same 5 P0–P1 rats). Spontaneous events covered a cortical area with an average diameter of 387.9 ± 8.3 µm (n = 317 events recorded in 5 P0–P1 rats) (Supplementary Movie S1), which was not significantly different from the evoked responses (320.2 ± 11.5 µm, n = 15 events in the same 5 P0–P1 rats). These results demonstrate that spontaneous events have very similar spatial properties when the sensory-evoked responses recorded with VSDI at this age. When the spontaneous events were superimposed on the predicted barrel field map of each individual animal, it became evident that 1) 76% of all spontaneous events (241 of 317 events recorded in 5 P0–P1 rats) were located in the barrel field and 2) the large majority of these spontaneous events were restricted to a small cortical region of 300–400 µm in diameter, which resembled in its localization and dimension a single-whisker-defined column (Fig. 2A1–4). Since discrete cortical barrels cannot be demonstrated with histological or immunocytochemical methods before P3 (Erzurumlu et al. 1990), we refer to these early neuronal networks as functional pre-barrel-related columns or functional pre-columns. In P0– P1 rats (n = 14 from 12 litters), 70–80% of the spontaneous events were localized in 1 or 2 pre-barrel-related columns and ∼20% in 3–6 pre-columns (Fig. 2A4–6,C). In this age group, the spontaneously occurring activity only rarely covered an area of >6 pre-barrel-related columns. In P6–P7 rats, spontaneous events (n = 462 in 15 animals from 11 litters) occurred every 2.6 ± 0.2 s (P < 0.05 vs. P0–P1), revealed an average duration at the half-maximal amplitude of 184 ± 6.6 ms (P < 0.001), and 77% of them were located in the barrel field (Fig. 2B1–3). As in P0–P1 rats, ∼70% of the spontaneous events recorded in P6–P7 animals covered only 1 or 2 barrelrelated columns (Fig. 2B4). About 20% of the events were localized in 3–6 columns and ∼10% in >6 barrel-related columns (Fig. 2B5–6,C and Supplementary Movie S2). These results demonstrate that 1) in the newborn (P0–P1) rat barrel cortex spontaneous events are predominantly restricted to single pre-barrel-related columns, 2) these spontaneous events have similar properties as sensory-evoked cortical responses, and 3) at the end of the first postnatal week spontaneously occurring activity may spread from the initially activated pre-barrel-related column to numerous neighboring columns.

electrophysiological responses depicted the topographic cortical representation of the 5 single arc 2 whiskers, namely A2 to E2 (Fig. 3A3). Similar to the evoked response recorded in the C2 barrel, single-whisker stimulation of the A2, B2, D2, or E2 whisker also elicited a local VSDI and electrophysiological response that was organized in a topographic manner. These experiments also demonstrated a close correlation of the FP responses with the VSDI responses in their location and spatial extent. The previous electrophysiological studies in both the newborn rat barrel cortex demonstrated 2 main types of spontaneously occurring and stimulus-evoked network activity patterns: 1) short-lasting gamma oscillations and 2) longer spindle bursts with a frequency of ∼10 Hz (Minlebaev et al. 2007; Yang et al. 2009). Our VSDI and simultaneous multichannel extracellular recordings demonstrate that both types of electrophysiological activity patterns contribute to the optical signal (Fig. 3B1). Following mechanical stimulation of 1304 Early columnar Network Oscillations in vivo

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a single whisker, the early FP response consisted of a short lasting burst in the gamma-frequency band (20–80 Hz; Wang and Buzsáki 1996; Lahtinen et al. 2002) and was correlated with the rising phase of the optical signal, indicating that the gamma activity plays an important role in synchronizing the pre-column network. This initial gamma activity was followed by a typical spindle burst as described previously (Minlebaev et al. 2007, 2009; Yang et al. 2009). Although in 11% of the experiments (n = 7 of 65 events recorded in 13 P0–P1 rats from 12 litters), a pure gamma burst could be observed, in most cases, the gamma activity was followed by a spindle burst (n = 56). Only 2 of 65 events (3%) recorded in P0–P1 rats were pure spindle bursts without the gamma activity. Both the spectrogram and the autocorrelation analyses revealed a clear gamma activity in the early response and the characteristic ∼10-Hz frequency in the subsequent spindle burst (Fig. 3B2,3). When the peak-to-peak amplitude of the evoked response was plotted against its distance to the center


Figure 3. Gamma and spindle bursts synchronize functional cortical columns. (A) The simultaneous VSDI and the multi-electrode recording of stimulus evoked the local cortical responses. The schematic illustration of the experimental set-up combining selective single-whisker stimulation, VSDI, and cortical 32-channel recordings using an 8-shank electrode (S1–S8) with shank spacing of 200 µm (A1). Mechanical stimulation of the C2 whisker in a P1 rat elicits a local VSDI (left) and local electrophysiological (right) response in the C2 barrel (A2). Color-coded localization of the evoked cortical VSDI (left) and electrophysiological (right) responses to stimulation of single whiskers in arc 2 (A3). The first negative peak amplitude of the electrophysiological response corresponds to the size of the color-coded circles as shown below the graph. Ten times baseline standard deviation was used as the threshold (∼150 µV) to define the spatial extent of the electrical response. (B) VSDI and FP response recorded simultaneously in the cortical E2 barrel following mechanical stimulation of the E2 whisker in a P1 rat. The corresponding spectrogram plot of the evoked FP response and autocorrelograms of early- and late-evoked FP response are shown below. Note the the early response with a period of ∼20 ms (arrowheads in B1–3) corresponding to a ∼50-Hz gamma burst discharge, while the late component showed a slower frequency. (C) The relationship between the peak-to-peak amplitude and the distance from the center of the ( pre-) barrel-related column for evoked gamma bursts (left) and spindle bursts (right) in P0–P1 (upper panels, n = 44 recordings in 4 animals from 4 litters) and P6–P7 rats (lower panels, n = 59 recordings in 5 animals from 4 litters).

Thalamic Activity Elicits Local Cortical Bursts In order to study the functional role of the thalamus in the generation of sensory-evoked cortical oscillations, we performed multi-channel extracellular recordings in the VPM nucleus of the thalamus in newborn rats in vivo (Fig. 5A1). The tracks of the DiI-labeled electrodes were subsequently reconstructed in histological sections to allow identification of the thalamic recording sites (Fig. 5A2). As in the barrel cortex, single-whisker stimulation elicited a local thalamic FP response that was accompanied by MUA (Fig. 5A3) and which consisted of an early brief component with the gamma activity and a slower late response (Fig. 5A4). In all recordings from P0 to P1 rats, the early gamma activity in the FP responses correlated with the local MUA (Fig. 5B). This correlation could clearly be demonstrated in the average power spectrum and coherence analyses of the evoked FP and MUA responses, which revealed a peak at 40–50 Hz (n = 10 P0–P1 rats from 7 litters) (Fig. 5C). These data demonstrate that the thalamus generates gamma burst activity in the newborn rat in vivo upon sensory stimulation. In order to study the relation between sensory-evoked thalamic burst activity and cortical gamma bursts, we performed simultaneous multi-channel extracellular recordings in both the VPM nucleus of the thalamus and barrel cortex (Fig. 6A). From a total of 1088 thalamic and 544 neocortical recording sites in 29 P0–P1 rats from 22 litters, we found upon mechanical single-whisker stimulation only 14 functional thalamocortical connections showing clear gamma bursts in both the thalamus and cortex. This low connectivity rate can be explained by the fact that in contrast to the cortical VSDI-guided recordings, the thalamic multi-electrode recording does not allow an a priori identification of the whisker representation. The transfer of sensory information from the VPM thalamus to the barrel cortex was surprisingly reliable already in P0–P1

rats, showing no failures (data not shown). In P0–P1 rats, the early thalamic gamma response had a stimulus-to-onset latency of 36.7 ± 1.7 ms, a duration of 92.3 ± 2.7 ms and was followed by a late response of 710.8 ± 53.6 ms in duration (n = 14 connections in 13 animals) (Fig. 6B). The early cortical gamma burst had an onset latency of 56.1 ± 2.5 ms, a duration of 89.1 ± 3.7 ms and was followed by a spindle burst of 644.5 ± 44.5 ms in duration (n = 14 connections in 13 animals). The thalamocortical transfer in P0–P1 rats required 19.3 ± 0.9 ms, which was also evident in the cross-correlogram of the early MUA responses (Fig. 6C1). Furthermore, the early response showed significant coherence between the thalamus and the cortex in the gamma frequency range (Fig. 6C2). Further, the proof for the role of the thalamus in generating the cortical gamma activity in newborn rats came from experiments in which the VPM was locally activated by bipolar electrical stimulation and the resulting cortical response was recorded with a multi-channel electrode. The thalamic stimulation site was identified by responses to single-whisker stimulation in simultaneous 32- and 16-channel recordings in both the thalamus and barrel cortex, respectively (Fig. 7A1). When the thalamic recording site showing the maximal response to whisker deflection was activated via bipolar electrical stimulation, a cortical response very similar to the sensory-evoked response could be observed in the corresponding pre-barrel-related column (Fig. 7A2,B). The duration (84.9 ± 3.8 ms) and frequency (37.7 ± 2.1 Hz) of the cortical gamma bursts evoked by barreloid electrical stimulation was not significantly different compared with the sensory-evoked gamma bursts (89.3 ± 8.6 ms and 41.8 ± 3.2 Hz, respectively, n = 7 P1 rats from 7 litters). These results demonstrate that electrical stimulation of the thalamus is capable of eliciting a cortical response that resembles the cortical activity pattern evoked by mechanical stimulation of a single whisker. In contrast, bipolar electrical stimulation of a site located 400 µm above or 200 µm aside of the barreloid elicited no clear response at any of the 16 cortical recording sites (Fig. 7A3), demonstrating that the stimulation protocol activated only a small region of thalamic tissue. Our data demonstrate that 1) the thalamocortical projection reliably transfers the sensory information from the whisker to the barrel cortex as early as P0–P1, 2) thalamic gamma bursts are capable of driving local gamma bursts in the barrel cortex following single-whisker stimulation in P0–P1 rats, and 3) the thalamic activity is sufficient to trigger local gamma bursts in the barrel cortex.

The Thalamus Is Important for the Generation of Spontaneously Occurring Cortical Bursts Next we addressed the question whether the local spontaneously occurring activity observed in the barrel cortex of P0–P1 rats with VSDI (Fig. 2) requires the thalamic activity or whether it is generated independently from the thalamus by intracortical mechanisms. The average occurrence of spontaneous cortical spindle bursts (gamma-containing and pure spindle bursts grouped together) was 0.9 ± 0.2 min−1 (n = 6 in P0–P1), which is ∼35% of the results of Minlebaev et al. (2007) (2.5 ± 0.4 min−1 in P2–P7 rats) and Marcano-Reik et al. (2010) (∼40/15 min, equal to 2.7 min−1 in P2–P6 rats). The lower occurrence of spindle bursts in our data might be due to age differences, since we focused on the P0–P1 age group. Cerebral Cortex June 2013, V 23 N 6 1305


of the barrel as identified by VSDI, it became evident that gamma bursts in P0–P1 rats are spatially confined to an area of ∼400 µm in diameter, whereas spindle bursts cover larger areas of ∼600 µm in diameter (upper panels in Fig. 3C). In agreement with our previous VSDI data, the spatial extent of the electrophysiological responses became larger with increasing age (lower panels in Fig. 3C). When in P0–P1 rats, the stimulus-evoked cortical FP response was compared with the corresponding MUA, a clear relationship between both signals could be detected (Fig. 4A). This correlation became more evident in the average power spectrum and coherence analyses of the evoked FP responses and MUA (n = 56 events recorded in 9 P0–P1 rats from 8 litters). The averaged FP and MUA response of the gamma burst revealed a clear peak at the frequency of 40–50 Hz, which was also evident in the coherence plot of the FP versus MUA (Fig. 4B1). A correlation between FP and MUA could also be demonstrated for the spindle bursts, which revealed a peak at the frequency of ∼10 Hz (Fig. 4B2). These results demonstrate that 1) gamma and spindle bursts are the electrophysiological activity patterns underlying the local VSDI signals evoked by single-whisker stimulation, 2) short-lasting gamma activity precedes the spindle burst, 3) gamma bursts in P0–P1 rats synchronize a pre-barrel-related column of ∼400 µm in diameter, and 4) the neuronal discharges correlate with the synchronized FP responses.


Figure 4. Cortical bursts correlate with MUA. (A) FP and multi-unit recordings of single-whisker-evoked responses in the barrel cortex. The schematic illustration of the experimental set-up with a 4-shank 16-channel electrode (A1). FP (upper traces) and MUA responses (lower traces) to single-whisker stimulation recorded in a P1 rat with the electrodes shown in panel 1 (A2). Average (black trace) and superimposed 20 single (shaded area) cortical FP responses and corresponding poststimulus time histogram (PSTH) of 20 succeeding whisker stimulations every 20 s (A3). Note the presence of gamma activity in the early response in the FP and MUA. (B) Average spectrum and coherence analyses of evoked gamma burst (B1) and spindle burst (B2) responses recorded in 9 P0–P1 rats. Black traces show averages and the shaded area represents the 95% CI. Note the prominent 40–50 Hz activity in the averaged gamma burst (filled arrowheads) and ∼10 Hz peak in the spindle burst response (open arrowheads). Shaded areas in (B) represent 95% CI.


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Downloaded from http://cercor.oxfordjournals.org/ at UB Mainz on July 3, 2013 Figure 5. Whisker stimulation elicits gamma activity in the thalamus. (A) Electrophysiological recordings of the whisker-evoked FP and MUA responses in the thalamic VPM of the newborn rat. The schematic illustration of the experimental set-up with a 4-shank 32-channel electrode located in the thalamus (A1). The schematic illustration of a sagittal brain section showing the location of the recording electrodes in the VPM thalamus (left picture taken from Paxinos and Watson 1998) (A2). The image of cytochrome oxidase stained section from the newborn rat showing 3 DiI-stained electrode tracks S1, S3, and S4 (right). Representative thalamic responses to single-whisker stimulation recorded with multi-electrode S1–S4 in a P0 rat (A3). The enlargement of response recorded at channel 24 with the corresponding spectrogram plot (A4). Note the gamma component in early response (arrowhead in A4). (B) Average (blue trace) and 20 superimposed single (shaded area) FP responses with corresponding poststimulus time histogram (PSTH) of 20 succeeding whisker stimulations every 30 s in a P1 rat. (C) Average spectrum and coherence analyses of evoked early thalamic responses recorded in 10 P0–P1 rats. Note the prominent gamma activity in the averaged FP and MUA spectrum and in the coherence plot (arrowheads). Shaded areas in (C) represent 95% CIs.

In all 14 simultaneous recordings with a functional thalamocortical connection from 13 P0–P1 rats (12 litters), we observed spontaneously occurring activity (Fig. 8A).

Spontaneously occurring activity in the barrel cortex consisted only in 52% of all events (n = 146 of 280 events) of an initial gamma component followed by a spindle burst Cerebral Cortex June 2013, V 23 N 6 1307


Figure 6. Whisker stimulation evoked cortical gamma bursts are preceded by the local thalamic activity. (A) Simultaneous multi-channel recordings of sensory-evoked responses to single-E5-whisker stimulation in both the VPM thalamus and barrel cortex of a P1 rat. The schematic illustration of the experimental set-up with a 4-shank 32-channel electrode located in the thalamus (blue) and a 4-shank 16-channel electrode in the cortex (black). The images show electrode tracks of the 4 DiI-stained electrodes S1–S4 in the thalamus. (B) Whisker stimulation evoked gamma and spindle burst response recorded simultaneously with cortical channel 2 and thalamic channel 27 in the P1 rat. (C) Cross-correlation of the MUA (C1) and coherence analysis (C2) of 20 evoked FP responses recorded simultaneously in both the thalamus and barrel cortex. Note that the thalamic MUA precedes cortical MUA (C1) and that the FP coherence plot reveals a clear gamma component (arrowhead in C2). Shaded area in C2 represents 95% CI.

(Fig. 8A). Twenty percent (57 of 280 events) showed only the initial gamma component and 28% (77 of 280 events) were pure spindle bursts without any obvious gamma activity, in contrast to the stimulus-evoked responses (11% pure gamma burst and 3% pure spindle bursts). Similar to the VSDI data, the duration of spontaneously occurring activity was also shorter than that of evoked responses (496.1 ± 19 ms, n = 280 events and 735.8 ± 17.3 ms, n = 140 events, respectively, P < 0.001). All spontaneous cortical events (n = 280) were preceded by the thalamic activity (Fig. 8B). 1308 Early columnar Network Oscillations in vivo

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Next we focused on those spontaneous events containing gamma bursts. The power spectrum analyses of the spontaneous gamma burst activity in both the cortex and thalamus showed a prominent peak at 30–40 Hz in FP and MUA recordings (Fig. 8C1,2). The synchronization of spikes in this frequency band is also evident in the coherence plot of FP versus MUA recordings in both the cortex and the thalamus (Fig. 8D). Finally, the promi-nent FP coherence between the cortex and thalamus at 30–40 Hz (Fig. 8E2) is also similar between spontan-eously occurring and whisker stimulationinduced gamma bursts.

Downloaded from http://cercor.oxfordjournals.org/ at UB Mainz on July 3, 2013 Figure 7. Local electrical stimulation of the thalamus elicits cortical gamma and spindle bursts. (A) Simultaneous multi-channel recordings of the thalamic (blue traces) and cortical responses (black traces) to B1-whisker stimulation in a P1 rat (A1). The schematic illustration of the experimental set-up with a 4-shank 32-channel electrode located in the thalamus (blue) and a 4-shank 16-channel electrode in the barrel cortex (black) is shown in the upper right panel. Note that the thalamic MUA responses to B2-whisker stimulation are located at channels 22 and 23. Bipolar electrical stimulation (120 μA, 100 μs duration) of the B1 thalamic barreloid at channels 22 and 23 elicits the local cortical responses in the B1 barrel (A2). No cortical responses were elicited when the electrical stimulation was performed outside the B1 thalamic barreloid (A3). (B) The cortical responses recorded at electrode number 10 to B1-whisker stimulation (B1) and thalamic B1 barreloid (B2) electrical stimulation. Both responses reveal a prominent gamma component in spectrogram and FP spectrum analysis (marked by the arrowheads). Note the antidromic population spike (arrowhead) upon thalamic stimulation. Shaded areas in right panels of (B) represent 95% CI. Cerebral Cortex June 2013, V 23 N 6 1309


Figure 8. The spontaneous cortical gamma activity arises in the thalamus. (A) Simultaneous 40 s recording of spontaneously occurring activity in the barrel cortex and VPM thalamus of a P1 rat. Upper trace represents the cortical FP recording; cortical (black bars) and thalamic MUA (blue bars) are presented below (A1). The 3 spontaneous events i–iii marked in A1 are shown at higher resolution with corresponding spectrograms (A2). Note the gamma activity in (i) and (iii). An averaged spectrogram of 140 evoked (black) and 140 spontaneous (red) cortical gamma bursts in 14 simultaneous recordings with a functional thalamocortical connection from 13 P0–P1 rats (A3). Shaded areas represent 95% CI. (B) Average (black trace) and 24 superimposed single (shaded area) spontaneous FP events and MUA recorded simultaneously in the cortex (B1) and in the thalamus (B2). Thalamic and cortical recordings were aligned to the onset of the cortical spontaneous FP events. (C) Spectrum analyses of the cortical (C1) and thalamic (C2) spontaneous FP and MUA events shown in (B). (D) Coherence analyses of spontaneous FP events versus MUA recorded simultaneously in the cortex (D1) and thalamus (D2). (E) Cross-correlation of the spontaneous MUA (E1) and coherence analysis of the FP (E2) recorded simultaneously in the thalamus and barrel cortex. Arrowheads in (C–E) point to the gamma component. Diagrams in (C–E) were calculated from the data marked by red dashed line in (B). Shaded areas in (C–E) represent 95% CI.


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bursts may originate in the barrel cortex itself, in adjacent cortical areas or in non-thalamic subcortical structures. Modulation of Thalamic Gamma Bursts by Cortico-Thalamic Feedback In order to study a potential cortico-thalamic feedback modulation of the thalamic network activity, the cortex of P0–P1 rats was inactivated by the surface application of 1% lidocaine. Mechanical stimulation of a single whisker elicited the typical response in the VPM and barrel cortex (Fig. 10A1). After lidocaine application, the cortical responses were, as expected, completely blocked (Fig. 10A2), while the thalamic activity could be clearly seen. However, in all 7 animals from 6 litters, the cortical inactivation desynchronized the activity in the corresponding barreloid. No obvious gamma peak was observed in the thalamic MUA spectrograms (Fig. 10A2) and the level of evoked MUA was significantly (P < 0.05) reduced to 82.3 ± 5% (n = 7, Fig. 10A3). Accordingly, the power of the gamma component in the thalamic response was significantly (P < 0.001) reduced to 57.2 ± 3.9% (n = 7) when compared with the control responses obtained before cortical inactivation (Fig. 10A3). Similar results could be observed for the spontaneously occurring activity (Fig. 10B). After lidocaine application to the cortex, the occurrence of spontaneous thalamic bursts did not change significantly (0.93 ± 0.15 vs. 0.88 ± 0.18 min−1, n = 6 P1 rats). However, the level of thalamic MUA within a burst was reduced to 64.1 ± 14.2% (n = 6) and the power of the gamma component of these spontaneous thalamic bursts was significantly (n = 6, P < 0.01) reduced to 55.2 ± 7.3%, when compared with the controls (Fig. 10B3). These data indicate that already in P0–P1 rats, the cortex influences the properties of gamma burst activity in the thalamus, suggesting a cortical modulatory influence on the thalamic activity via cortico-thalamic feedback circuits at very early developmental stages.

Discussion We show here for the first time that the rat barrel cortex in vivo reveals a precise topographic and functional columnar organization already at the day of birth. Although the graded expression of several transcription factors sets the rudimentary topography of thalamocortical connectivity, thereby specifying cortical areas and creating crude cortical maps during early prenatal stages (López-Bendito and Molnár 2003; O’Leary and Sahara 2008), our data indicate that spontaneously occurring and whisker stimulation-induced burst activity plays an important role in the formation of functional cortical columnar networks already before the cortex has gained its 6-layered structure. We demonstrate that gamma bursts synchronize local cortical networks into functional ontogenetic columns of 300–400 µm in diameter in the rat barrel cortex already at the day of birth and that the thalamus plays a central role in the generation of this cortical activity. Our data further indicate that a cortico-thalamic feedback loop modulates the thalamic spontaneously occurring and whisker stimulation-induced gamma burst activity already during neonatal stages of corticogenesis. An important finding of our study is the precise cortical representation of single whiskers already in the first 2 postnatal days, even before all cortical layers have been formed— Cerebral Cortex June 2013, V 23 N 6 1311


However, the spontaneously occurring gamma bursts recorded in both the thalamus and cortex of P0–P1 rats differed from the whisker stimulation-induced responses obtained at the same recording sites. The thalamic spontaneously occurring activity had a longer duration (134.9 ± 6.1 ms, P < 0.001, n = 14 connections in 13 P0–P1 rats, paired t-test) and slower frequency (33.4 ± 1.3 Hz, P < 0.001) when compared with the sensory-evoked thalamic responses (92.3 ± 3.9 ms and 40.2 ± 1.7 Hz, respectively). Cortical spontaneous gamma bursts also had a longer duration of 120.1 ± 4.8 ms (P < 0.001) and a slower frequency of 32.3 ± 1.1 Hz (P < 0.01) when compared with the sensory-evoked cortical responses (89.1 ± 5.5 ms and 39.1 ± 2.3 Hz, respectively) (Fig. 8A3). Finally, the thalamocortical transfer of spontaneously occurring activity was slower (36.2 ± 2 ms) than that of sensory-evoked responses (19.3 ± 1.1 ms, P < 0.001), as it is also evident from the MUA crosscorrelation (Fig. 8E1). However, despite these differences, these data demonstrate that spontaneous gamma bursts in the cortex are closely linked to the thalamic activity patterns. To directly address the question, whether the thalamus plays a critical role in generating spontaneous cortical bursts, we analyzed the functional consequences of acute local inactivation of the thalamus on the expression of sensory-evoked and spontaneous cortical bursts recorded in the barrel cortex of P0–P1 rats. Simultaneous multi-electrode recordings in both the VPM and barrel cortex upon single-whisker stimulation (control in Fig. 9A1) were used to determine the site of thalamic lesion. Since the thalamic high-density recordings allowed the unequivocal identification of the activated barreloid (cf. Fig. 7A1), the thalamic representation of the stimulated whisker could be locally inactivated by an electrolytic lesion (Fig. 9A4). Following this local thalamic lesioning, single-whisker stimulation elicited, as expected, no longer any kind of activity in the thalamus and only small sensory-evoked responses in the cortex (Fig. 9A2). When compared with the cortical MUA responses obtained before the local thalamic inactivation, the average number of spikes and the power of the gamma spectrum in the early response, which corresponds to the gamma burst phase, were significantly reduced to 11.4 ± 5.3% and 8.2 ± 4.6%, respectively (n = 6 P0–P1 rats from 6 litters, Fig. 9A3). In addition, the number of spikes and the power of the MUA spectrum (